Biosynthesis Of Salidroside Through Transglycosylation Mediated By β-glucosidase In Non-aqueous Medium

Salidroside is a kind of compounds of special physiological function, with wide range of applications because of its advantages such as anti-aging, anti-tumor, anti-fatigue, cardiovascular protection and so on. At present, the extraction from plants and chemical synthesis are the main sources of obtaining salidroside. Due to the low yield in plant extraction, as well as the complicated synthesis steps and pollution to the environment in chemical synthesis, the scope of application of salidroside is severely limited. Fortunately, owing to the several advantages such as high stereoselectivity, mild reaction conditions and high purity of products, the enzymatic biotransformation of salidroside has been the focus in synthesis of salidroside.In this paper, β-glucosidase catalytic transglycosylation in non-aqeous medium was studied. The results showed that the best glycosyl donor is alicin, and phosphate buffer/acetonitrile (1:9) is the optimal reaction medium. When the molar ratio of salicin and p-hydroxyphenylethanol was 1:2, and at pH 5.5,50 ℃,225 r/min, after the reaction of 30 h, the product salidroside was up to a maximum concentration of 22.30 g/L. Under the same condition, compared to the direct glycosylation in the non-aqueous medium catalyzed by P-glucosidase, the transglycosylation obtaining higher concentration of salidroside, as well as the shorter reation time required to reach equilibrium.The enzymatic properties of the cross-link P-glucosidase aggregates (CLGAs) and magnetic cross-link β-glucosidase aggregates (M-CLGAs) in the organic solvent system were studied. In the biosynthesis of salidroside through transglycosylation catalyzed by CLGAs and M-CLGAs in the above organic solvent system, the reaction temperature, rotation speed, reaction time, the operation stability and kinetic parameters of the enzymes were probed. The experiments results of CLGAs showed that in the organic solvent system, the optimum pH and temperature were 6.0 and 60 ℃, as well as its Vmax and Km were 2.59 μmoL·min-1·mg-1 and 28.36 mmoL/L, respectively. When the transglycosylation carried out for 72 h, the product salidroside concentration reached a maximum of 29.80 g/L. Furthermore, after the four batches of reaction (288 h), the residual relative activity of CLGAs declined from 84% to 51.30%. Likewise, the experiments results of M-CLGAs showed that in the oranic solvent system, the optimum pH and temperature were 6.0 and 60 ℃, as well as its Vmax and Km were 2.85 μmoL·min-1·mg-1 and 26.39 mmol/L.When the transglycosylation carried out for 72h, the product concentration reached a maximum of 27.40 g/L. And after the four batches of reaction (288 h), the residual relative activity of CLGAs declined from 80% to 53.67%. For the results of enzyme operation stability, it demonstrated that the CLGAs and M-CLGAs are stable in the organic solvent in transglycosylation for biosynthesis of salidroside.